Daniel:Notebook/ComboLock/2016-10-12
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Padlock0301 Test (Started Friday 10-7)[edit]
Testing the amplification using the forward (AmpF-CL-v2) and primer24 reverse set, as well as some dilutions.
Protocol[edit]
- qPCR
- Make the following master mixes
- Set up qPCR reactions according to table in next section
- Aliquot 48 uL correct master mix into each well
- Add 2 uL sample to appropriate well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 48 uL TBE, 12 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Master Mix | nfH20 | AmpF-CL-v2 | Reverse Primer | Rev. Primer Conc. (uM) | Amt. Reverse Primer | Kapa SYBR Fast MM | Total |
MM1 | 90.2 | 2.05 | primer24 | 100 | 2.05 | 102.5 | 196.8 |
MM2 | 93.48 | 0.41 | AmpR6.3-27 | 100 | 0.41 | 102.5 | 196.8 |
MM3 | 90.2 | 2.05 | AmpR6.3-28 | 10 | 2.05 | 102.5 | 196.8 |
MM4 | 93.48 | 0.41 | AmpR6.3-29 | 10 | 0.41 | 102.5 | 196.8 |
qPCR Table[edit]
Sample | Molecule Count | Lane | Reverse Primer | Reverse Primer Conc. (uM) | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | Reverse Primer | 100 uM AmpF | H2O | Total Volume (uL) | Master Mix |
Sample 0A | 10 pmol | A1 | primer24 | 100 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM1 |
Sample 0B | 10 pmol | A2 | primer24 | 100 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM1 |
Sample 0C | 10 pmol | A3 | primer24 | 100 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM1 |
NTC (rev) | 0 | A4 | primer24 | 100 | 0 | 25 | 0.5 | 0.5 | 24 | 50 | MM1 |
Sample 0A | 10 pmol | A1 | AmpR6.3-27 | 100 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 0B | 10 pmol | A2 | AmpR6.3-27 | 100 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 0C | 10 pmol | A3 | AmpR6.3-27 | 100 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
NTC (rev) | 0 | A4 | AmpR6.3-27 | 100 | 0 | 25 | 0.1 | 0.1 | 24 | 50 | MM2 |
Sample 0A | 10 pmol | A1 | AmpR6.3-28 | 10 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 0B | 10 pmol | A2 | AmpR6.3-28 | 10 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 0C | 10 pmol | A3 | AmpR6.3-28 | 10 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
NTC (rev) | 0 | A4 | AmpR6.3-28 | 10 | 0 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 0A | 10 pmol | A1 | AmpR6.3-29 | 10 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
Sample 0B | 10 pmol | A2 | AmpR6.3-29 | 10 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
Sample 0C | 10 pmol | A3 | AmpR6.3-29 | 10 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
NTC (rev) | 0 | A4 | AmpR6.3-29 | 10 | 0 | 25 | 0.1 | 0.1 | 24 | 50 | MM4 |
Results[edit]
- 2016-10-12-qPCR-Padlock0301-PrimerTest.png
qPCR
- 2016-10-12-Padlock0301-PrimerTest-Gel1.png
Gel Image 1
- 2016-10-12-Padlock0301-PrimerTest-Gel2.png
Gel Image 2
Library Prep[edit]
- qPCR
- Make the following master mix
- 71.75 uL nfH2O
- 2.05 uL AmpF-CL-6.4
- 102.5 uL Kapa SYBR Fast
- Aliquot 43 uL master mix into each well
- Prep samples according to following table
- Add 2 uL sample to appropriate well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- Size Select Gel
- Mix 20 uL sample and 4 uL 6x loading dye into separate tubes for each sample
- Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
- Aliquot 24 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Image in gel doc
- 2016-10-12-Padlock0301-SizeSelect.png
Before image
- 2016-10-12-Padlock0301-SizeSelect-After.png
After image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate overnight with vigorous shaking; continued tomorrow
Sample | Template Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF-CL-v2 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1A | 10 pmol | A1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 10 pmol | B1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 10 pmol | C1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | D1 | 25 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
File:20161012-qpCR-Padlock0301-LibraryPrep.png